A slipped replication intermediate model is stabilized by the syn orientation of N-2-aminofluorene- and N-2-(acetyl)aminofluorene-modified guanine at a mutational hotspot

Debjani Roy, Brian E. Hingerty, Robert Shapiro, Suse Broyde

Research output: Contribution to journalArticle

Abstract

The Escherichia coli NarI restriction enzyme recognition site 5'G 1G 2C 3G 4C 5C 63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G 4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 41474151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G 4 by the related aromatic amine 2- aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G 4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' 3' C 11 G 10 G 9 T 8 5' Intermediate II: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' C 11 G 10 ········ G 9 T 8 5' Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G 4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.

Original languageEnglish (US)
Pages (from-to)1301-1311
Number of pages11
JournalChemical Research in Toxicology
Volume11
Issue number11
DOIs
StatePublished - 1998

Fingerprint

2-Acetylaminofluorene
Guanine
Elongation
Frameshift Mutation
Escherichia coli
Amines
Molecular mechanics
2-aminofluorene
DNA
Potential energy
Mechanics
Conformations
Carcinogenesis
Plasmids
Enzymes

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

A slipped replication intermediate model is stabilized by the syn orientation of N-2-aminofluorene- and N-2-(acetyl)aminofluorene-modified guanine at a mutational hotspot. / Roy, Debjani; Hingerty, Brian E.; Shapiro, Robert; Broyde, Suse.

In: Chemical Research in Toxicology, Vol. 11, No. 11, 1998, p. 1301-1311.

Research output: Contribution to journalArticle

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abstract = "The Escherichia coli NarI restriction enzyme recognition site 5'G 1G 2C 3G 4C 5C 63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G 4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 41474151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G 4 by the related aromatic amine 2- aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G 4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' 3' C 11 G 10 G 9 T 8 5' Intermediate II: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' C 11 G 10 ········ G 9 T 8 5' Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G 4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.",
author = "Debjani Roy and Hingerty, {Brian E.} and Robert Shapiro and Suse Broyde",
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AU - Shapiro, Robert

AU - Broyde, Suse

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N2 - The Escherichia coli NarI restriction enzyme recognition site 5'G 1G 2C 3G 4C 5C 63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G 4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 41474151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G 4 by the related aromatic amine 2- aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G 4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' 3' C 11 G 10 G 9 T 8 5' Intermediate II: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' C 11 G 10 ········ G 9 T 8 5' Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G 4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.

AB - The Escherichia coli NarI restriction enzyme recognition site 5'G 1G 2C 3G 4C 5C 63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G 4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 41474151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G 4 by the related aromatic amine 2- aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G 4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' 3' C 11 G 10 G 9 T 8 5' Intermediate II: 5' G 1 G 2 C 3 G 4 C 5 C 6 A 7 3' C 11 G 10 ········ G 9 T 8 5' Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G 4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.

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